The present invention relates to sequences that encode dimethylallyl-diphosphate:L-tryptophan dimethylallyltransferase (xe2x80x9cDmaWxe2x80x9d or xe2x80x9cdimethylallyltryptophan synthasexe2x80x9d), an enzyme present in some grass endophytes, and that catalyzes the formation of 4- xcex3,xcex3-dimethylallyltryptophan. This enzyme activity is the first committed step in the production of ergot alkaloids, including those with clavine and ergoline-ring structures. Such alkaloids include clavines, lysergic acid, lysergic acid amides, and ergopeptines. The sequences encode a DmaW from fungi that are symbionts of commercially significant grasses.
Certain fungal species exist as symbiotic and integral parts of grasses and are passed from generation to generation of plants, but many are not passed from plant to plant except by transmitting in seeds of maternal plant lineages. Representatives of these fungi are the Neotyphodium species (xe2x80x9cNeotyphodiumxe2x80x9d and sometimes xe2x80x9cAcremoniumxe2x80x9d, for example, N. coenophialum) and Epichloe species (e.g. E. festucae and E. typhina), which are symbionts and integral parts of many grass cultivars. These fungi, termed xe2x80x9cendophytesxe2x80x9d, are seed-transmissible at extremely high efficiency. Their symbioses with host grasses are characterized by mutual benefits to the hosts and symbionts. Benefits to the grass hosts include protection from insects and vertebrates, and resistance to water stress (drought). Anti-insect activities are mainly due to pyrrolopyrazine and pyrrolizidine alkaloids produced by the endophytes. Anti-vertebrate activities are mainly due to indole alkaloids, including the ergot alkaloids (clavines, lysergic acid and its derivatives, and ergopeptines).
Tall fescue is grown on over 14 million hectares as an important forage, turf and conservation grass; most of the tall fescue grown in the U.S. contains ergot-alkaloid-producing endophytes. The anti vertebrate activity of the ergot alkaloids, which manifests as xe2x80x9ctall fescue toxicosisxe2x80x9d in cattle and other livestock, causes losses estimated at more than $600 million per year.
In 1992, Gebler and Poulter purified the DmaW enzyme from Claviceps sp. ATCC 26245 to a single protein band observable by SDS-PAGE electrophoresis, and fragmented the protein with CNBr. Gebler et al., 114 Journal of the American Chemical Society 7354 (1992). The three resulting fragments were purified and their N-termini sequenced. In research by one of the inventors of the present invention, there was disclosed a sequence of a dmaW gene (herein dmaW) from C. fusiformis ATCC 26245 organism from which the sequence was identified was mis-named C. purpurea by the supplier to the ATCC, and was actually C. fusiformis.) Tsai et al., 216 Biochem and Biophys Res Comm 119 (1995). The C. fusiformis sequence from that research is 58% identical to the present sequences at the DNA level. More recently, a C. purpurea dmaW sequence was disclosed in Tudzynski et al., 261 Molec Gen Genet 133 (1999), and is 62% identical at the DNA level to the present sequence.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. For example, in some instances above, the publication was less than one year before the filing date of this patent application. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant at the time of filing, and does not constitute an admission as to the accuracy of the dates or contents of these documents.
It is therefore an object of the present invention to provide sequences useful to engineer ergot alkaloid-deficient symbionts, thus ergot-alkaloid-deficient plants.
It is a further object to provide methods to engineer ergot alkaloid-deficient endophytes.
It is yet another object to provide ergot alkaloid-deficient seeds.
It is yet another object to provide plants with ergot alkaloid-deficient endophytes.
It is also an object of the invention to provide materials such as vectors for engineering ergot alkaloid-deficient endophytes.
It is also an object of the invention to provide enzymes useful in ergot alkaloid synthesis.
Also, it is an object to use the present nucleic acid compounds to determine the potential or lack of potential of symbiont strains to produce ergot alkaloids.
Other objects will be apparent from the present disclosure.
Definitions:
For the purposes of the present application, the following terms have the following meanings. All other terms have the meaning as specifically recognized in the art.
xe2x80x9cAllelic variantxe2x80x9d is meant to refer to a full length gene or partial sequence of a full length gene that occurs at essentially the same locus (or loci) as the referent sequence, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants can also comprise alterations in the 5xe2x80x2 or 3xe2x80x2 untranslated regions of the gene (e.g., in regulatory control regions).
xe2x80x9cFragmentxe2x80x9d is meant to refer to any subset of the referent nucleic acid sequence.
xe2x80x9cKnockout constructxe2x80x9d means a DNA sequence which has been altered via any known means, for example, deletion, insertion, point mutation or rearrangement, so as to alter or eliminate the function of the naturally-occurring sequence product, but not so as to alter the ability of the DNA sequence to recombine with the naturally-occurring sequence.
xe2x80x9cKnockout mutantsxe2x80x9d are cells, embryos, fungi or plants in which a naturally-occurring dmaW sequence has been replaced through genetic engineering with a knockout construct, so as to result in a ergot alkaloid-deficient phenotype, especially a dimethylallyl-diphosphate:L-tryptophan dimethylallyltransferase- deficient phenotype.
xe2x80x9cProteinsxe2x80x9d means any compounds which comprise amino acids, including peptides, polypeptides, fusion proteins, etc.
Moreover, for the purposes of the present invention, the term xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d entity refers to one or more of that entity; for example, xe2x80x9ca proteinxe2x80x9d or xe2x80x9ca nucleic acid moleculexe2x80x9d refers to one or more of those compounds or at least one compound. As such, the terms xe2x80x9caxe2x80x9d (or xe2x80x9canxe2x80x9d), xe2x80x9cone or morexe2x80x9d and xe2x80x9cat least onexe2x80x9d can be used interchangeably herein. It is also to be noted that the terms xe2x80x9ccomprisingxe2x80x9d, xe2x80x9cincludingxe2x80x9d, and xe2x80x9chavingxe2x80x9d can be used interchangeably. Furthermore, a compound xe2x80x9cselected from the group consisting ofxe2x80x9d refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure, protein or nucleic acid molecule is a compound that has been removed from its natural milieu. As such, xe2x80x9cisolatedxe2x80x9d and xe2x80x9cbiologically purexe2x80x9d do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis.